Neuropeptides and their use for pest control

ABSTRACT

The present invention discloses novel pest control compounds comprising NPF polypeptides and methods for using such compounds in the control of pests.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application is a continuation-in-part of U.S.application Ser. No. 09/295,849, filed Apr. 21, 1999.

BACKGROUND OF THE INVENTION

[0002] Many blood-ingesting pests are known to feed on humans andanimals, and many pests are vectors for pathogenic microorganisms whichthreaten human and animal health, including commercially importantlivestock, pets and other animals. Various species of mosquitoes, forexample, transmit diseases caused by viruses, and many are vectors fordisease-causing nematodes and protozoa. Mosquitoes of the genusAnopheles transmit Plasmodium, the protozoan which causes malaria, adevastating disease which results in approximately 1 million deathsannually. The mosquito species Aedes aegypti transmits an arbovirus thatcauses yellow fever in humans. Other arboviruses transmitted by Aedesspecies include the causative agents of dengue fever, eastern andwestern encephalitis, Venezuelan equine encephalitis, St. Louisencephalitis, chikungunya, oroponehe and bunyarnidera. The genus Culex,which includes the common house mosquito C. pipiens, is implicated inthe transmission of various forms of encephalitis and filarial worms.The common house mosquito also transmits Wuchereria bancrofti and Brugiamalayi, which cause various forms of lymphatic filariasis, includingelephantiasis. Trypanasoma cruzi, the causative agent of Chagas'disease, is transmitted by various species of blood-ingestingTriatominae bugs. The tsetse fly (Glossina spp.) transmits Africantrypanosomal diseases of humans and cattle. Many other diseases aretransmitted by various blood-ingesting pest species. The order Dipteracontains a large number of blood-ingesting and disease-bearing pests,including, for example, mosquitoes, black flies, no-see-ums (punkies),horse flies, deer flies and tsetse flies.

[0003] Various pesticides have been employed in efforts to control oreradicate populations of disease-bearing pests, such as disease-bearingblood-ingesting pests. For example, DDT, a chlorinated hydrocarbon, hasbeen used in attempts to eradicate malaria-bearing mosquitoes throughoutthe world. Other examples of chlorinated hydrocarbons are BHC, lindane,chlorobenzilate, methoxychlor, and the cyclodienes (e.g., aldrin,dieldrin, chlordane, heptachlor, and endrin). The long-term stability ofmany of these pesticides and their tendency to bioaccumulate render themparticularly dangerous to many non-pest organisms.

[0004] Another common class of pesticides is the organophosphates, whichis perhaps the largest and most versatile class of pesticides.Organophosphates include, for example, parathion, MALATHION, diazinon,naled, methyl parathion, and dichlorvos. Organophosphates are generallymuch more toxic than the chlorinated hydrocarbons. Their pesticidaleffect results from their ability to inhibit the enzyme cholinesterase,an essential enzyme in the functioning of the insect nervous system.However, they also have toxic effects on many animals, including humans.

[0005] The carbamates, a relatively new group of pesticides, includesuch compounds as carbamyl, methomyl, and carbofuran. These compoundsare rapidly detoxified and eliminated from animal tissues. Theirtoxicity is thought to involve a mechanism similar to the mechanism ofthe organophosphates; consequently, they exhibit similar shortcomings,including animal toxicity.

[0006] A major problem in pest control results from the capability ofmany species to develop pesticide resistance. Resistance results fromthe selection of naturally-occurring mutants possessing biochemical,physiological or behavioristic factors that enable the pests to toleratethe pesticide. Species of Anopheles mosquitoes, for example, have beenknown to develop resistance to DDT and dieldrin. DDT substitutes, suchas MALATHION, propoxur and fenitrothion are available; however, the costof these substitutes is much greater than the cost of DDT.

[0007] There is clearly a longstanding need in the art for pesticidalcompounds that are pest-specific, that reduce or eliminate direct and/orindirect threats to human health posed by presently availablepesticides, that are environmentally compatible in the sense that theyare biodegradable, and are not toxic to non-pest organisms, and havereduced or no tendency to bioaccummulate.

[0008] Many pests, including for example blood-inbibing pests, mustconsume and digest a proteinaceous meal to acquire sufficient essentialamino acids for growth, development and the production of mature eggs.Adult pests, such as adult mosquitoes, need these essential amino acidsfor the production of vitellogenins by the fat body. These vitellogeninsare precursors to yolk proteins which are critical components ofoogenesis. Many pests, such as house flies and mosquitoes, produceoostatic hormones that inhibit egg development by inhibiting digestionof the protein meal, and thereby limiting the availability of theessential amino acids necessary for egg development.

[0009] Serine esterases such as trypsin and trypsin-like enzymes(collectively referred to herein as “TTLE”) are important components ofthe digestion of proteins by insects. In the mosquito, Aedes aegypti, anearly trypsin that is found in the midgut of newly emerged females isreplaced, following the blood meal, by a late trypsin. A female mosquitotypically weighs about 2 mg and produces 4 to 6 μg of trypsin withinseveral hours after ingesting blood meal. Continuous biosynthesis atthis rate would exhaust the available metabolic energy of a femalemosquito; as a result, the mosquito would be unable to produce matureeggs, or even to find an oviposition site. To conserve metabolic energy,the mosquito regulates TTLE biosynthesis with a peptide hormone namedTrypsin Modulating Oostatic Factor (TMOF). Mosquitoes produce TMOF inthe follicular epithelium of the ovary 12-35 hours after a blood meal;TMOF is then released into the hemolymph where it binds to a specificreceptor on the midgut epithelial cells, signaling the termination ofTTLE biosynthesis.

[0010] This regulatory mechanism is not unique for mosquitoes; fleshflies, fleas, sand flies, house flies, dog flies and other pests whichingest protein as part of their diet have similar regulatory mechanisms.

[0011] In 1985, Borovsky purified an oostatic hormone 7,000-fold anddisclosed that injection of a hormone preparation into the body cavityof blood imbibed mosquitoes caused inhibition of egg development andsterility (Borovsky, D. [1985] Arch. Insect Biochem. Physiol.2:333-349). Following these observations, Borovsky (Borovsky, D. [1988]Arch. Ins. Biochem. Physiol. 7:187-210) reported that injection orpassage of a peptide hormone preparation into mosquitoes inhibited theTTLE biosynthesis in the epithelial cells of the gut. This inhibitioncaused inefficient digestion of the blood meal and a reduction in theavailability of essential amino acids translocated by the hemolymph,resulting in arrested egg development in the treated insect. Borovskyobserved that this inhibition of egg development does not occur when theoostatic hormone peptides are inside the lumen of the gut or other partsof the digestive system (Borovsky, D. [1988], supra).

[0012] Following the 1985 report, the isolated hormone, (a ten aminoacid peptide) and two TMOF analogues were disclosed in U.S. Pat. Nos.5,011,909 and 5,130,253, and in a 1990 publication (Borovsky, et al.[1990] FASEB J 4:3015-3020). Additionally, U.S. Pat. No. 5,358,934discloses truncated forms of the full length TMOF which have prolinesremoved from the carboxy terminus, including the peptides YDPAP, YDPAPP,YDPAPPP, and YDPAPPPP.

[0013] Neuropeptides Y (NPY) are an abundant family of peptides that arewidely distributed in the central nervous system of vertebrates. NPYpeptides have also been recently isolated and identified in a cestode, aturbellarian, and in terrestrial and marine molluscs (Maule et al., 1991“Neuropeptide F: A Novel Parasitic Flatworm Regulatory Peptide fromMoniezia expansa (Cestoda: Cyclophylidea)” Parasitology 102:309-316;Curry et al., 1992 “Neuropeptide F: Primary Structure from theTurbellarian, Arthioposthia triangulata” Comp. Biochem. Physiol.101C:269-274; Leung et al., 1992 “The Primary Structure of NeuropeptideF (NPF) from the Garden Snail, Helix aspersa” Regul. Pep. 41:71-81;Rajpara et al., 1992 “Identification and Molecular Cloning ofNeuropeptide Y Homolog that Produces Prolonged Inhibition in AplysiaNeurons” Neuron. 9:505-513).

[0014] Invertebrate NPYs are highly homologous to vertebrate NPYs. Themajor difference between vertebrate and invertebrate NPYs occurs at theC-terminus where the vertebrate NPY has an amidated tyrosine (Y) whereasinvertebrates have an amidated phenylalanine (F). Because of thisdifference, the invertebrate peptides are referred to as NPF peptides.

[0015] Cytoimmunochemical analyses of NPY peptides suggest that they areconcentrated in the brain of various insects, including the Coloradopotato beetle Leptinotarsa decemlineata (Verhaert et al., 1985 “DistinctLocalization of FMRFamide- and Bovine Pancreatic Polypeptide-LikeMaterial in the Brain, Retrocerebal Complex and Subesophageal Ganglionof the Cockroach Periplaneta americana” L. Brain Res. 348:331-338;Veenstra et al., 1985 “Immunocytochemical Localization of PeptidergicNeurons and Neurosecretory Cells in the Neuro-Endocrine System of theColorado Potato Beetle with Antisera to Vertebrate Regulatory Peptides”Histochemistry 82:9-18). Partial purification of NPY peptides in insectssuggests that both NPY and NPF are synthesized in insects (Duve et al.,1981 “Isolation and Partial Characterization of PancreaticPolypeptide-like Material in the Brain of the Blowfly alliphoravomitoria” Biochem. J. 197, 767-770).

[0016] Researchers have recently isolated two neuropeptides withNPF-like immunoreactivity from brain extracts of the Colorado potatobeetle. The researchers purified the peptides using C₁₈ reversed phasehigh-pressure liquid chromatography (HPLC), and determined theirstructure using mass spectrometry. The deduced structures of thesepeptides are: Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ IDNO. 1) and Ala-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 2)designated NPF I and NPF II, respectively (Spittaels, Kurt, PeterVerhaert, Chris Shaw, Richard N. Johnston et al. [1996] Insect Biochem.Molec. Biol. 26(4):375-382).

BRIEF SUMMARY OF THE INVENTION

[0017] The subject invention pertains to materials and methods forcontrolling pests. In a preferred embodiment, the subject inventioninvolves the use of a polypeptide comprising an NPF peptide to controlpests (referred to herein as the “NPF polypeptides”). Specificallyexemplified are NPF polypeptides comprising an amino acid sequenceselected from the group consisting ofAla-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 1) andAla-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 2) and correspondingnon-amidated NPF polypeptides Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe(SEQ ID NO. 3) and Ala-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 4),compositions comprising such NPF polypeptides and methods for using suchcompounds and pesticidal compositions. In a preferred mode, the NPFpolypeptides comprise an amino acid sequence which consists of a nativeNPF peptide or a fragment, analogue, derivative or other functionalequivalent of an NPF peptide.

[0018] Further exemplified NPF polypeptides include those comprising anamino acid sequence selected from the group consisting ofArg-Pro-Pro-Thr-Arg-Phe-Arg-Phe-amide (SEQ ID NO. 5),Arg-Pro-Pro-Thr-Arg-Phe-Arg-Phe (SEQ ID NO. 6),Ala-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 7),Ala-Pro-Gln-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 8),Ala-Asn-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 9),Ala-Asn-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 10),Ala-Asp-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 11),Ala-Asp-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 12),Pro-Ile-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 13),Pro-Ile-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 14),Ala-Gln-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 15),Ala-Gln-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 16),Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 17),Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 18),Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 19),Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 20), Leu-Arg-Leu-Arg-Phe-amide(SEQ ID NO. 21), Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 22), Arg-Pro-Pro-Thr(SEQ ID NO. 23), and Arg-Phe-Arg-Phe (SEQ ID NO. 24). NPF polypeptidesof SEQ ID NOs. 5, 7, 9, 11, 13, and 15 are native NPF peptides frommosquito, American cockroach, or fruitfly, and are homologous with theamino acid sequences of SEQ ID NOs. 1 and 2, which are native to theColorado potato beetle. NPF polypeptides of SEQ ID NOs. 6, 8, 10, 12,14, and 16 are the respective non-amidated versions of these native NPFpeptides. Specifically exemplified NPF analogues are those comprising anamino acid sequence selected from the group consisting of SEQ ID NOs.17-24.

[0019] The NPF polypeptides of the subject invention are particularlyactive against blood-ingesting pests, e.g., species of mosquitoes suchas Aedes aegypti, which are vectors of many arthropod-borne viraldiseases (arboviruses). These pests utilize serine esterases, such asTTLE as their primary blood digesting enzymes.

[0020] One aspect of the subject invention pertains to methods forcontrolling blood-ingesting pests by applying to a pest or to apest-inhabited locus, a pesticidal formulation comprising (a) an NPFpolypeptide comprising a native NPF peptide or functional equivalentthereof and (b) a pesticidally effective carrier.

[0021] The subject invention further pertains to the use of NPFpolypeptides to control other pests, including non-blood ingestingagricultural pests. These pests include, for example, coleopterans(beetles), lepidopterans (caterpillars), and mites. The compounds of thesubject invention can also be used to control household pests including,but not limited to, ants and cockroaches.

[0022] The present invention also includes addition salts, complexes andprodrugs such as esters of the NPF polypeptides, especially the nontoxicpharmaceutically or agriculturally acceptable acid addition salts. Theacid addition salts can be prepared in standard manner in a suitablesolvent from the parent compound and an excess of an acid, such ashydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic,succinic, ethanedisulfonic or methanesulfonic acids. Also, theN-terminus and C-terminus of the NPF polypeptides can be chemicallymodified to further inhibit proteolysis by metabolic enzymes, forexample, by N-terminal carboxylation and/or C-terminal amidation.

[0023] Dextrorotory amino acids can also be usefully employed in the NPFpolypeptides of the present invention to inhibit the ability ofproteases to degrade the peptides of the subject invention.

[0024] NPF polypeptides in which only conservative substitutions havebeen made are also provided by the present invention. Analogues of theNPF polypeptides which have one or more amino acid substitutions forminga branched peptide (e.g., by substitution with an amino acid or aminoacid analogue having a free amino- or carboy-side chain that forms apeptide bond with a sequence of one or more amino acids, including butnot limited to prolines) or allowing circularization of the peptide(e.g., by substitution with a cysteine, or insertion of a cysteine atthe amino- or carboxy-terminus or internally, to provide a sulfhydrylgroup for disulfide bond formation), are also provided.

[0025] Also, derivation of the NPF polypeptides with long chainhydrocarbons facilitates passage through the cuticle into the pest bodycavity. Therefore, a further embodiment of the subject inventionpertains to compositions comprising the NPF polypeptides bound to lipidsor other carriers.

[0026] Analogues and derivatives, and other functional equivalents,included within the scope of the invention are those which retain someor all of the pesticidal activity of native NPF peptides, or those whichshow improved activity as compared to a corresponding native NPFpeptide. Thus, included within the scope of the invention arepesticidally active native NPF peptides, fragments, analogues (e.g.,homologues), derivatives, or other functional equivalents of native NPFpeptides.

[0027] Yet another aspect of the subject invention pertains to DNAsequences encoding the peptides of the subject invention disclosedherein. These DNA sequences can be readily synthesized by a personskilled in the art. The sequences may be used to transform anappropriate host to confer upon that host the ability to express the NPFpolypeptides. Hosts of particular interest include bacteria, algae,yeasts, insect viruses, and plants. For each of these hosts, the DNAsequences may be specifically designed by a person skilled in the art toutilize codons known to be optimally expressed in the particular hosts.Advantageous promoters can also easily be utilized. Bacteria, yeasts,plants, algae, viruses, and other hosts each may be used to producepeptide for further use, or these hosts can be used as vehicles fordirect application of the peptide to the target pest. A plant speciescan be transformed to express the NPF polypeptides, resulting in a plantvariety that is toxic to a target pest species which feeds on the plant.Pest control is achieved when the pest ingests the transformed plantmaterial thereby exposing the pest to the NPF polypeptide. Methods fortransforming plant cells utilizing, for example Agrobacteria, are wellknown to those skilled in the art.

[0028] Another aspect of the subject invention pertains to a method ofcontrolling pests comprising administering to said pest an effectiveamount of one or more NPF polypeptides.

[0029] The subject invention provides pest control compositions whereinthe NPF polypeptides are formulated for application to the target pestsor their situs. In a specific embodiment, the present invention providesrecombinant hosts, which express an NPF polypeptide. The recombinanthost can be a procaryotic or eucaryotic cell, including for example,yeast or algae cells which are transformed to express an NPFpolypeptide. The transformed host may also be a virus. The transformedhost can be applied to a pest's habitat, (e.g., where the target pest isa mosquito, the transformed host can be applied to a body of water whichserves as a habitat for mosquito larvae), where the pest will ingest thetransformed host resulting in control of the pest by the NPFpolypeptide.

[0030] The methods and materials of the subject invention provide anovel approach to controlling pests and pest-transmitted diseases.

[0031] As used herein, the term “pesticidally effective” is used toindicate an amount or concentration of a pesticide, e.g., an NPFpolypeptide, which is sufficient to reduce the number of pests in ageographical locus, as compared to a corresponding geographical locus inthe absence of the amount or concentration of the pesticide.

[0032] The term “pesticidal” is not intended to refer only to theability to kill pests, but also includes the ability to interfere with apest's life cycle in any way that results in an overall reduction in thepest population. For example, the term “pesticidal” includes inhibitionor elimination of reproductive ability of a pest, as well as inhibitionof a pest from progressing from one form to a more mature form, e.g.,transition between various larval instars or transition from larvae topupa or pupa to adult. Further, the term “pesticidal” is intended torefer to all phases of a pest life cycle; thus, for example, the termincludes larvicidal, ovicidal and adulticidal action.

[0033] The word “transform” is broadly used herein to refer tointroduction of an exogenous polynucleotide sequence into a prokaryoticor eukaryotic cell by any means known in the art (including for example,direct transmission of a polynucleotide sequence from a cell or virusparticle, transmission by infective virus particles, and transmission byany known polynucleotide-bearing substance) resulting in a permanent ortemporary alteration of genotype and in an immortal or non-immortal cellline.

[0034] The terms “peptide,” “polypeptide,” and “protein” as used hereinare intended to refer to amino acid sequences of any length.

[0035] Without intending to be bound by theory, the current invention isbased on the determination that NPF adversely affects TTLE biosynthesisin the midgut of female Aedes aegypti fed a blood meal and injected withNPF polypeptide. Because the structure of NPF is different from TMOF itappears that NPF does not bind to a TMOF-specific binding site on thegut receptor but to a different site on the same or different receptor.Furthermore, cytoimmunochemical analysis, by the inventors, of themosquito gut after the blood meal, using antiserum against NPF, hassurprisingly revealed exocrine cells with NPF-like molecules that aresynthesized by mosquito epithelial cells 24 hours after a blood meal.NPF therefore appears to be a secondary signal in a cascade of signals:first TMOF is released from the ovary, TMOF then binds to a TMOF gutreceptor (Borovsky et al. [1994] FASEB J. 8:350-355) that stimulates thesynthesis and release of NPF from gut specific exocrine cells. NPF thenbinds to a receptor site on the gut at a site which may be adjacent toor part of the TMOF receptor, resulting cessation of biosynthesis ofTTLE. This surprising discovery opens the door to a new generation ofNPF pesticides, which inhibit biosynthesis of TTLE in a more directmanner than previously disclosed TMOF peptides.

BRIEF DESCRIPTION OF THE SEQUENCES

[0036] SEQ ID NO. 1 is an amidated neuropeptide designated NPF I.

[0037] SEQ ID NO. 2 is an amidated neuropeptide designated NPF II.

[0038] SEQ ID NO. 3 is a non-amidated version of the NPF I peptide.

[0039] SEQ ID NO. 4 is a non-amidated version of the NPF II peptide.

[0040] SEQ ID NOs. 5-24 are amidated and non-amidated versions of NPFpolypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1 is a graph showing the inhibitory affect of NPF I ontrypsin biosynthesis when injected into whole mosquitos. NPF I resultedin a 50% inhibition of trypsin biosynthesis when injected at a 1×10⁻⁶ Mconcentration.

[0042]FIG. 2 is a graph showing the inhibitory affect of NPF II ontrypsin biosynthesis when injected into whole mosquitos. NPF II resultedin a 35% inhibition of trypsin biosynthesis at a 1×10⁻³ M concentration.

[0043]FIG. 3 is a graph showing the inhibitory affect of NPF I ontrypsin biosynthesis when injected into ligated mosquito abdomens. NPF Iresulted in a 54% inhibition of trypsin biosynthesis at a 1×10⁻⁶ Mconcentration. These results strongly suggest that NPF acts on areceptor in the gut and not through a cell signaling transductionpathway which originates in the brain.

DETAILED DISCLOSURE OF THE INVENTION

[0044] The subject invention concerns NPF polypeptides that can be usedto control target pests. Specifically exemplified is the use of NPFpolypeptides in controlling mosquitos and other pests. A method ofcontrolling pests is also specifically exemplified herein, which methodemploys the use of NPF I and/or NPF II, which have the sequencesAla-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 1) andAla-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 2), respectively, aswell as non-amidated versions of NPF I and NPF II,Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 3) andAla-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 4), respectively.

[0045] Further exemplified NPF polypeptides include those comprising anamino acid sequence selected from the group consisting ofArg-Pro-Pro-Thr-Arg-Phe-Arg-Phe-amide (SEQ ID NO. 5),Arg-Pro-Pro-Thr-Arg-Phe-Arg-Phe (SEQ ID NO. 6),Ala-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 7),Ala-Pro-Gln-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 8),Ala-Asn-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 9),Ala-Asn-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 10),Ala-Asp-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 11),Ala-Asp-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 12),Pro-Ile-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 13),Pro-Ile-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 14),Ala-Gln-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 15),Ala-Gln-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 16),Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 17),Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 18),Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 19),Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 20), Leu-Arg-Leu-Arg-Phe-amide(SEQ ID NO. 21), Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 22), Arg-Pro-Pro-Thr(SEQ ID NO. 23), and Arg-Phe-Arg-Phe (SEQ ID NO. 24). NPF polypeptidesof SEQ ID NOs. 5, 7, 9, 11, 13, and 15 are native NPF peptides frommosquito, American cockroach, or fruitfly, and exhibit homology with theamino acid sequences of SEQ ID NOs. 1 and 2, which are native to theColorado potato beetle. NPF polypeptides of SEQ ID NOs. 6, 8, 10, 12,14, and 16 are the respective non-amidated versions of these native NPFpeptides. Specifically exemplified NPF analogues are those comprising anamino acid sequence selected from the group consisting of SEQ ID NOs.17-24.

[0046] The term “pest” as used herein includes mosquitoes, insects andother organisms which adversely affect, humans, plants or animals,including, for example, organisms that remove blood, tissue and/or anyother fluid from their prey or host. Pests controlled according to thesubject invention specifically include those which regulate TTLEconcentrations in the gut by a mechanism which involves the binding of aligand to a receptor to trigger an increase or a decrease in thesynthesis of digestive enzymes, e.g., TMOF binding to its receptor.Examples of pests which can be controlled according to the subjectinvention include, but are not limited to, mosquitos, fleshflies, fleas,sandflies, houseflies, dogflies, and insects which attack plants.

[0047] The pest control compositions according to the subject inventioncomprise an NPF polypeptide, or a fragment, derivative, analogue orother functional equivalent of an NPF polypeptide, as a component, or asthe sole component. The pest control compositions may further comprise acarrier solution, compound, or molecule. Pest control compositions ofthe subject invention also include an NPF polypeptide, or a fragment,derivative, analogue or other functional equivalent of an NPFpolypeptide, contained in or associated with a cell, virus, plant, ormembrane. Examples include, but are not limited to, transformedbacteria, mammalian cells, algae, fungi, yeast viruses, or plants thatproduce an NPF polypeptide.

[0048] The term “functional equivalent” as used herein refers to apolypeptide sequence comprising full-length native NPF polypeptide, or acomprising fragment, analogue (e.g., homologue), or derivative of afull-length native NPF. Functional equivalents include, for example, anNPF polypeptide in salt, complex, analogue, or derivative form as wellas a fragment, derivative or analogue of a native NPF peptide, whichretains some or all of the biological activity of the native NPFpeptide.

[0049] The NPF polypeptides may be presented as fusion proteins orpeptides, the amino acid sequence of which includes one or more NPFpolypeptides of the present invention. In various specific embodiments,two or more of the NPF polypeptides are linked, for example, by peptidebonds between the N-terminus of one portion and the C-terminus ofanother portion. In other aspects, one or more of the NPF polypeptidescan be linked to one or more heterologous polypeptides to formpesticidal fusion peptides. Molecules comprising such portions linked byhydrocarbon linkages are also provided. Derivatives of the foregoingfusion proteins are also provided (e.g., branched, curcularized,N-terminal carboxylated or C-terminal amidated).

[0050] In one embodiment the fusion protein or peptide comprises arepeating unit of at least 4 amino acids (e.g., a multimer). There maybe, for example, from 2 to 10 or more repeating units. Preferably, therepeating unit is connected through at least one amino acid which iscleaved by a pest gut enzyme. Methods of recombinantly producingpeptides in cells as multimers are known in the art. (Rao et al., 1996“Synthesis and expression of genes encoding putative insect neuropeptideprecursors in tobacco,” Gene 175:1-5; Tortiglione et al., 1999 “NewGenes for Pest Control,” Genetics and Breeding for Crop Quality andResistance, 159-163). For example, a tandemly repeated DNA cassette forthe expression of NPF peptides can be constructed. As used herein, apest gut enzyme is an enzyme which is present in the gut of a pest.Preferably, the pest is a mosquito or a lepidopteran. In a specificembodiment, the repeating units are connected through an arginine.

[0051] Analogues which have one or more amino acid substitutions forminga branched peptide (e.g., by substitution with an amino acid or aminoacid analogue having a free amino- or carboxy-side chain that forms apeptide bond with a sequence of one or more amino acids, including butnot limited to prolines) or allowing circularization of the peptide(e.g., by substitution with a cysteine, or insertion of a cysteine atthe amino- or carboxy-terminus or internally, to provide a sulfhydrylgroup for disulfide bond formation), are also provided.

[0052] Nonclassical amino acids or chemical amino acid analogues canreplace existing amino acid residues of the NPF polypeptides or beinserted into the NPF polypeptides between existing amino acid residuesof the NPF polypeptides or added to a terminus of the NPF polypeptidesof the present invention. Non-classical amino acids include, but are notlimited to, the D-isomers of the common amino acids, 2,4-diaminobutyricacid, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyricacid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,τ-butylglycine, τ-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, C-methyl amino acids, N-methyl amino acids, and amino acidanalogues in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary). Dextrorotary amino acids are indicatedherein by a parenthetical D, i.e., “(D)”, immediately preceding thedextrorotary amino acid.

[0053] The NPF compounds include peptides containing, as a primary aminoacid sequence, all or part of an exemplified NPF polypeptide sequence.The NPF compounds thus include NPF polypeptides having conservativesubstitutions, i.e., altered sequences in which functionally equivalentamino acid residues are substituted for residues within the sequenceresulting in a peptide which is functionally active. For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid of a similar polarity which acts as a functionalequivalent, resulting in a silent alteration. In one aspect of thepresent invention, conservative substitutions for an amino acid withinthe sequence may be selected from other members of the class to whichthe amino acid belongs (see Table 1). Conservative substitutions alsoinclude substitutions by amino acids having chemically modified sidechains which do not eliminate the pesticidal properties of the resultingNPF compound. TABLE 1 Class of Amino Acid Examples of Amino AcidsNonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly,Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

[0054] In a specific embodiment, the subject invention is directedtoward a method of controlling blood-ingesting pests comprisingpreparing a treatment comprising an NPF compound and applying saidtreatment to said blood-ingesting pests. In another embodiment thesepeptides are used to control agricultural pests.

[0055] Preparation of novel pest control compounds. The NPF polypeptidesof the invention can be prepared by well-known synthetic procedures. Forexample, the polypeptides can be prepared by the well-known Merrifieldsolid support method. See Merrifield (1963) J. Amer. Chem. Soc.85:2149-2154 and Merrifield (1965) Science 150:178-185. This procedure,using many of the same chemical reactions and blocking groups ofclassical peptide synthesis, provides a growing peptide chain anchoredby its carboxyl terminus to a solid support, usually cross-linkedpolystyrene or styrenedivinylbenzene copolymer. This method convenientlysimplifies the number of procedural manipulations since removal of theexcess reagents at each step is effected simply by washing of thepolymer.

[0056] Alternatively, these peptides can be prepared by use ofwell-known molecular biology procedures. Polynucleotides, such as DNAsequences, encoding the NPF polypeptides of the invention can be readilysynthesized. Such polynucleotides are a further aspect of the presentinvention. These polynucleotides can be used to genetically engineereukaryotic or prokaryotic cells, for example, bacteria cells, insectcells, algae cells, plant cells, mammalian cells, yeast cells or fungicells for synthesis of the peptides of the invention. Viruses may alsobe genetically modified using such polynucleotides, to serve as vectorsfor the delivery of the polynucleotides to insect pests or to othercells. One example of a cell line usefully transformed according to theteachings of the present invention is the insect cell line Sf9(Spodoptera frugiperda), deposit number ATCC CRL 1711, available fromthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. 20852 USA. An example of a useful virus includes the Baculovirus,Autographa californica Nuclear Polyhedrosis Virus (AcNPV), which isavailable from Texas A&M University, Texas Agricultural ExperimentStation, College Station, Tex. 77843, and described in Smithand Summers(1978) Virology 89:517-527; and (1979) J. Virology 30:828-838. Othernuclear polyhedrosis viruses (See World Health Organization TechnicalReport No. 531) such as Spodoptera frugiperda (Sf MNPV), Choristoneurafumiferana (Cf MNPV) (Smithand Summers [1981] J. Virol. 39:125-137), orSpodoptera littoralis (S1 NPV) (Harrap, et al. [1977] Virology 79:14-31)can be used instead of Autographa californica NPV. Other insect celllines can also be substituted for Spodoptera frugiperda (Sf9), forexample, Trichoplusia ni (Volkman and Summers [1975] J. Virol.16:1630-1637), Spodoptera exigua, Choristoneura fumiferana (Smith, andSummers [1981] J. Virol. 39:125-137) and Spodoptera littoralis (Harrap,K. A. et al. [1977] Virology 79:14-31).

[0057] In yet another embodiment, the subject invention is directed topolynucleotides which encode the subject NPF polypeptides.Polynucleotides can be produced by routine methods known in the art.[See S. L. Beaucage and M. H. Caruthers (1981), Tetrahedran Lett.22:1859].

[0058] The polynucleotides of the present invention can be amplifiedusing Polymerase Chain Reaction (PCR), a repetitive, enzymatic, primedsynthesis of a nucleic acid sequence. This procedure is well known andcommonly used by those skilled in this art (see Mullis, U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159; Saiki, et al. [1985] “EnzymaticAmplification of β-Globin Genomic Sequences and Restriction SiteAnalysis for Diagnosis of Sickle Cell Anemia,” Science 230:1350-1354.).PCR employs the enzymatic amplification of a DNA fragment of interestthat is flanked by two oligonucleotide primers that hybridize toopposite strands of the target sequence. The primers are oriented withthe 3′ ends pointing towards each other. Repeated cycles of heatdenaturation of the template, annealing of the primers to theircomplementary sequences, and extension of the annealed primers with aDNA polymerase result in the amplification of the segment defined by the5′ ends of the PCR primers. Since the extension product of each primercan serve as a template another primer, each cycle essentially doublesthe amount of DNA fragment produced in the previous cycle, resulting inthe exponential accumulation of the specific target fragment, up toseveral million-fold in a few hours. By using a thermostable DNApolymerase such as Taq polymerase, which is isolated from thethermophilic bacterium Thermus aquaticus, the amplification process canbe completely automated. Other useful enzymes are known to those skilledin the art.

[0059] PCR primers can be designed from the DNA sequences of the subjectinvention. In performing PCR amplification, a certain degree of mismatchcan be tolerated between primer and template. Therefore, mutations,deletions, and insertions (especially additions of nucleotides to the 5′end) of the exemplified sequences fall within the scope of the subjectinvention. These PCR primers can be used to amplify genes of interestfrom a sample providing another method for identifying andcharacterizing polynucleotide sequences encoding the NPF polypeptides.

[0060] The various methods employed in the preparation of the plasmidsand transformation of host organisms are well known in the art and aredescribed, for example, in U.S. Pat. Nos. 5,011,909 and 5,130,253. Thesepatents are incorporated herein by reference. These procedures are alsodescribed in Maniatis, et al. (1982) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York. Thus, it is within theskill of those in the genetic engineering art to extract DNA from itssource, perform restrictions enzyme digestions, electrophorese DNAfragments, tail and anneal plasmid and insert DNA, ligate DNA, transformcells, e.g., E. coli or plant cells, prepare plasmid DNA, electrophoreseproteins, and sequence DNA.

[0061] Production of recombinant hosts. In another embodiment, thesubject invention is directed to a cell transformed with apolynucleotide encoding a pesticidal NPF polypeptide. Hosts, which maybe employed according to techniques well known in the art for theproduction of the NPF polypeptides of the present invention, includeunicellular microorganisms, such as prokaryotes, i.e., bacteria; andeukaryotes, such as fungi, (including yeasts), algae, protozoa, molds,and the like, as well as plant cells, both in culture or in planta, andanimal cells. Specific bacteria which are susceptible to transformationinclude members of the Enterobacteriaceae, such as strains ofEscherichia coli; Salmonella; Bacillaceae, such as Bacillus subtilis;Pseudomonas; Pneumococcus; Streptococcus; Haemophilus influenzae, andyeasts such as Saccharomyces, among others.

[0062] In one embodiment of the present invention, the transformed hostis Bacillus sphaericus, which is known to be highly specific for controlof mosquito larvae. In a preferred embodiment, the host is Bacillussphaericus, serotype H5a5b, available from Abbott Laboratories asVectoLex CG Biological Larvicide (EPA Reg. No. 275-77).

[0063] The polynucleotide sequences of the subject invention can beintroduced directly into the genome of the transformable host cell orcan first be incorporated into a vector which is then introduced intothe host. Exemplary methods of incorporation include transduction byrecombinant phage or cosmids, transfection where specially treated hostbacterial cells can be caused to take up naked phage chromosomes, andtransformation by calcium precipitation. These methods are well known inthe art. Exemplary vectors include plasmids, cosmids, and phages.

[0064] It is well known in the art that when synthesizing a gene forimproved expression in a host cell it is desirable to design the genesuch that its frequency of codon usage approaches the frequency ofpreferred codon usage of the host cell. For purposes of the subjectinvention, “frequency of preferred codon usage” refers to the preferenceexhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. To determine the frequency of usage of aparticular codon in a gene, the number of occurrences of that codon inthe gene is divided by the total number of occurrences of all codonsspecifying the same amino acid in the gene. Similarly, the frequency ofpreferred codon usage exhibited by a host cell can be calculated byaveraging frequency of preferred codon usage in a large number of genesexpressed by the host cell. It is preferable that this analysis belimited to genes that are highly expressed by the host cell.

[0065] Thus, in one embodiment of the subject invention, bacteria,algae, fungi, yeast, plants, or other cells can be geneticallyengineered, e.g., transformed with polynucleotides encoding the subjectpeptides to attain desired expression levels of the subject peptides. Toprovide genes having enhanced expression, the DNA sequence of the genecan be modified to comprise codons preferred by highly expressed genesto attain an A+T content in nucleotide base composition which issubstantially that found in the transformed host cell. It is alsopreferable to form an initiation sequence optimal for the host cell, andto eliminate sequences that cause destabilization, inappropriatepolyadenylation, degradation and termination of RNA and to avoidsequences that constitute secondary structure hairpins and RNA splicesites. For example, in synthetic genes, the codons used to specify agiven amino acid can be selected with regard to the distributionfrequency of codon usage employed in highly expressed genes in the hostcell to specify that amino acid. As is appreciated by those skilled inthe art, the distribution frequency of codon usage utilized in thesynthetic gene is a determinant of the level of expression.

[0066] Assembly of the polynucleotide sequences of this invention can beperformed using standard technology known in the art. For example, astructural gene designed for enhanced expression in a host cell can beassembled within a DNA vector from chemically synthesizedoligonucleotide duplex segments. Preferably, the DNA vector or constructhas an operable promoter and suitable termination signals. Thepolynucleotide sequence can be introduced into a host cell and expressedby means known in the art. Preferably, the NPF compound produced uponexpression of the nucleotide sequence is functionally equivalent to thecorresponding purified NPF polypeptide. The present invention alsocomprises expression cassettes comprising the polynucleotides of thepresent invention and a expression vectors comprising polynucleotidesand/or expression cassettes of the present invention. According to thesubject invention, “functionally equivalent” indicates retention offunction such as, for example, pest control activity.

[0067] The present invention also includes chimeric polypeptidescomprising one or more heterologous polypeptides joined to one or moreNPF polypeptides, and also includes chimeric polypeptides comprising twoor more NPF polypeptides joined together. The portions which arecombined need not, themselves, be pesticidal so long as the combinationof portions creates a chimeric protein which is pesticidal. The chimerictoxins may include portions from toxins which do not necessarily actupon the TMOF receptor including, for example, toxins from Bacillusthuringiensis (B.t.). B.t. toxins and their various toxin domains arewell known to those skilled in the art. Preferred toxins originate withvarious strains of B.t. including, for example, B.t. israeliensis, B.t.tenebrionis, B.t. san diego, B.t. aizawai, B.t. subtoxicus, B.t. alesti,B.t. gallaeriae, B.t. sotto, B.t. kurstaki, B.t. berliner, B.t.tolworthi, B.t. dendrolimus and B.t. thuringiensis, and other B.t.toxins known in the art such as the various delta-endotoxins describedin U.S. Pat. No. 5,686,069.

[0068] With the teachings provided herein, one skilled in the art canreadily produce and use the various compounds and polynucleotidesequences described herein.

[0069] The polynucleotide sequences and compounds useful according tothe subject invention include not only the exemplified sequences butalso fragments of these sequences, variants, mutants, and fusionproteins which retain the characteristic pesticidal activity of thepeptides specifically exemplified herein. As used herein, the terms“variants” or “variations” of genes refer to polynucleotides havingdifferent nucleotide sequences but encoding the same polypeptides orencoding equivalent peptides having pesticidal activity. As used herein,the term “equivalent” in reference to a peptide or polypeptide refers tocompounds exhibiting some or all of the biological activity of nativeNPF peptides.

[0070] Variations of genes may be readily constructed using standardtechniques for making point mutations. Also, fragments of these genescan be made using commercially available exonucleases or endonucleasesaccording to standard procedures. For example, enzymes such as thenuclease, BAL31, or site-directed mutagenesis can be used tosystematically excise nucleotides from the ends of the genes. Also,genes which encode active fragments may be obtained using a variety ofrestriction enzymes. Proteases may be used to directly obtain activefragments of these peptides.

[0071] Polynucleotide sequences encoding NPF polypeptides can beintroduced into a wide variety of microbial or plant hosts with theresult that expression of the gene results, directly or indirectly, inthe production and maintenance of the NPF polypeptides. With suitablemicrobial hosts, e.g., yeast, Chlorella, the microbes can be applied tothe situs of the pest, where they will proliferate and be ingested bypest organisms, resulting in control of the pest. Alternatively, themicrobe hosting the gene can be killed and may optionally be treatedunder conditions that prolong the activity of the toxin and stabilizethe cell. Such killed cells can be applied to the habitat and/or to thehost or prey of the target pest. In one embodiment, the microbial orother host is transformed such that the gene encoding the pesticidal NPFpolypeptide is only exressed or maintained for a relatively short periodof time, such as days or weeks, so that the expression of the NPFpolypeptide does not continue indefinitely.

[0072] A wide variety of methods are available for introducing apolynucleotide sequence encoding a pesticidal polypeptide into amicroorganism host under conditions which allow for stable maintenanceand expression of the gene. These methods are well known to thoseskilled in the art and include, for example, the methods described inU.S. Pat. No. 5,135,867, which is incorporated herein by reference.

[0073] Synthetic genes which encode peptides which are functionallyequivalent to the NPF polypeptide of the subject invention can also beused to transform hosts. Methods for the production of synthetic genescan be found in, for example, U.S. Pat. No. 5,380,831.

[0074] Recombinant cells expressing a pest control compound can betreated to prolong the pesticidal activity of the NPF polypeptide andstabilize the cell. For example, such cells can be formulated as apesticide microcapsule comprising the NPF polypeptide within astabilized cellular structure that protects the toxin when themicrocapsule is applied to the environment of the target pest. Suitablehost cells include prokaryotes and eukaryotes. Preferred hosts includeprokaryotes and lower eukaryotes, such as algae and fungi. Therecombinant cell will preferably be intact and be substantially in theproliferative form when treated, rather than in a spore form.

[0075] Treatment of the microbial cell, e.g., a microbe containing thepolynucleotide sequence encoding the pesticidal polypeptide, can be bychemical or physical means, or by a combination of chemical and/orphysical means, so long as the technique does not completely diminishthe properties of the toxin, nor diminish the cellular capability ofprotecting the toxin. Methods for treatment of microbial cells aredisclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which areincorporated herein by reference.

[0076] Methods and formulations for control of pests. Control of pestsusing the NPF polypeptides of the subject invention can be accomplishedby a variety of methods known to those skilled in the art. These methodsinclude, for example, the application of recombinant microbes to thepests (or their locations), and the transformation of plants with genes(polynucleotide sequences) encoding the NPF polypeptides of the subjectinvention. Transformations can be made by those skilled in the art usingstandard techniques. Materials necessary for these transformations aredisclosed herein or are otherwise readily available to the skilledartisan.

[0077] The plant pests which can be controlled by the compounds of thesubject invention generally belong to the phylum Arthropoda, includingpests of the orders Coleoptera, Lepidoptera, Hemiptera and Thysanoptera.Other pests which can be controlled according to the subject inventioninclude members of the orders Diptera, Siphonaptera, Hymenoptera andPhthiraptera. Pests of the class Arachnida, such as ticks, mites, andspiders, can also be controlled by the NPF polypeptides of the presentinvention.

[0078] The use of the compounds of the subject invention to controlpests can be accomplished readily by those skilled in the art having thebenefit of the instant disclosure. For example, the control compoundsmay be encapsulated, included in a granular form, solubilized in wateror other appropriate solvent, powdered, and included into anyappropriate formulation for direct application to the pest. In apreferred embodiment for the control of plant pests, plants may begenetically transformed to express the pest control compound such that apest feeding upon the plant will ingest the control compound and therebybe controlled.

[0079] Where the polynucleotide sequence is introduced via a suitablevector into a microbial host, and said host is applied to theenvironment in a living state, it is preferred that certain hostmicrobes be used. Microorganism hosts are selected which are known tooccupy the “phytosphere” (phylloplane, phyllosphere, rhizosphere, and/orrhizoplane) of one or more crops of interest or the situs where the pestproliferates. These microorganisms are selected so as to be capable ofsuccessfully competing in the particular environment (crop and otherinsect habitats) with the wild-type organisms, provide for stablemaintenance and expression of the gene expressing the polypeptidepesticide, and, desirably, provide for improved protection of thepesticide from environmental degradation and inactivation.

[0080] A large number of microorganisms are known to inhabit thephylloplane (the surface of the plant leaves) and/or the rhizosphere(the soil surrounding plant roots) of a wide variety of important crops.These microorganisms include bacteria, algae, and fungi. Preferredmicroorganisms include bacteria, e.g., genera Bacillus, Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium; andalgae, e.g., Chlorella. Of particular interest are such phytospherebacterial species as Pseudomonas syringae, Pseudomonas fluorescens,Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens,Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti,Alcaligenes entrophus, and Azotobacter vinlandii and Bacillusthurnigensis; and phytosphere yeast species such as Rhodotorula rubra,R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C.diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, andAureobasidium pollulans. Pigmented microorganisms are particularlypreferred.

[0081] Formulated bait granules containing an attractant and the NPFpolypeptides, or recombinant microbes comprising toxin-encodingpolynucleotide sequences, can be applied to the soil. Formulated productcan also be applied as a seed-coating or root treatment or total planttreatment at later stages of the crop cycle. Plant and soil treatmentsmay be employed as wettable powders, granules or dusts, by mixing withvarious inert materials, such as inorganic minerals (phyllosilicates,carbonates, sulfates, phosphates, and the like) or botanical materials(powdered corncobs, rice hulls, walnut shells, and the like). Theformulations may include spreader-sticker adjuvants, stabilizing agents,other pesticidal additives, or surfactants. Liquid formulations may beaqueous-based or non-aqueous and employed as foams, gels, suspensions,emulsifiable concentrates, or the like. The ingredients may includerheological agents, surfactants, emulsifiers, dispersants, or polymers.

[0082] As would be appreciated by a person skilled in the art, theconcentration of NPF polypeptide in the pesticidal formulations of thepresent invention will vary widely depending upon the nature of theparticular formulation, particularly whether it is a concentrate or tobe used directly, and on the potency of the NPF polypeptide(s) selected.The NPF polypeptide will be present in at least about 0.0001% by weightand may be 100% by weight. The dry formulations will have from about0.0001-95% by weight of the NPF polypeptide, while the liquidformulations will generally be from about 0.0001-60% by weight of thesolids in the liquid phase. The formulations that contain cells willgenerally have from about 1 to about 10⁴ cells/mg. These formulationswill be administered at about 50 mg (liquid or dry) to 1 kg or more perhectare.

[0083] The formulations can be applied to the environment of the pest,e.g., soil and foliage, by spraying, dusting, sprinkling, or the like,and/or to hosts or prey of the pests, e.g., plants, and humans or otheranimals.

[0084] In applications to the environment of the target pest, atransformant strain can be applied to the natural habitat of the pest.In some cases, the transformant strain will continue to grow in the pestupon ingestion and produce NPF polypeptide following ingestion by thepest. The transformed organism may be applied by spraying, soaking,injection into the soil, seed coating, seedling coating or spraying, orthe like. Where administered in the environment, concentrations of theorganism will generally be from 1 to 10¹⁰ cells/ml, and the volumeapplied per hectare will be generally from about 0.1 oz to 2 lbs ormore. Where administered to a plant part inhabited by the target pest,the concentration of the organism will usually be from 10³ to 10⁶cells/cm².

[0085] In aquatic environments, pest control may be attained at or belowthe surface by adjusting the specific gravity of the microbe. This canbe accomplished by, for example, varying the lipid content of thetransformant microorganism strain. It is known that indigenous aquaticalgae float due to their lipid content. A variation in lipid contentwill allow the transformant strain to be distributed at desired depthsbelow the water surface.

[0086] For commercial formulations, the organisms may be maintained in anutrient medium which maintains selectivity and results in a low rate ofproliferation. Various media may be used, such as yeast extract orL-broth. Once the organism is to be used in the field, thenon-proliferating concentrate may be introduced into an appropriateselective nutrient medium, grown to high concentration, generally fromabout 10⁵ to 10⁹ cells/ml and may then be employed for introduction intothe environment of the pest.

[0087] All of the U.S. patents and other references cited herein arehereby incorporated by reference, as are U.S. patent application Ser.No. 09/295,846, (UF-223) “Transformed Cells Useful for the Control ofPests”; U.S. patent application Ser. No. 09/551,737, (UF-223C1)“Transformed Cells Useful for the Control of Pests”; U.S. patentapplication Ser. No. 09/296,113, (UF-224) “Materials and Methods Usefulfor the Control of Insect Larvae”; U.S. patent application Ser. No.09/551,738, (UF-224C1) “Materials and Methods Useful for the Control ofInsect Larvae”; U.S. patent application Ser. No. 09/295,996, (UF-230)“Novel Peptides and the Use Thereof to Control Pests”; and U.S. patentapplication Ser. No. 09/295,924, (IPTL Docket No. 4137-120)“Compositions and Methods for Controlling Pests”.

[0088] Following are examples which illustrate procedures for practicingthe invention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Effect of NPF Polypeptides on Trypsin Biosynthesis

[0089] To find out if NPF I and II affect trypsin biosynthesis in themidgut of female Aedes aegypti, females were fed a blood meal andimmediately injected with 0.25 μl of the peptide at concentrations of2.5 μg to 12.5pg and 30 hours later the midguts were removed and assayedfor trypsin biosynthesis (Borovsky et al., 1990 “Mosquito OostaticFactor: A Novel Decapeptide Modulating Trypsin-Like Enzyme Biosynthesisin the Midgut” FASEB J. 4:3015-3020; Borovsky et al 1993 “MassSpectrometry and Characterization of Aedes aegypti Trypsin ModulatingOostatic Factor (TMOF) and its Analog” Insect Biochem. Molec. Biol.23:703-712). Each experiment was repeated 3 times (5 females per group)and the results are expressed as % inhibition of trypsinbiosynthesis±S.E.M. (FIG. 1). Fifty percent inhibition of trypsinbiosynthesis was achieved at a concentration of 10⁻⁶M NPF I. NPF II waseffective at a dose of 10⁻³M (78%±10), at 10⁻⁶M NPF II inhibited trypsinbiosynthesis by 35% (FIG. 2).

[0090] To determine if NPF I releases a neuroendocrine factor from thebrain or the thoracic ganglia which in turn may release TMOF from theovary, female Aedes aegypti were fed a blood meal, immediately ligatedand injected with different concentrations of NPF I(10⁻³M to 10⁻⁹M) in0.25 μl of sterile distilled water. Thirty hours later, abdomens wereremoved and 3 groups of 5 abdomens per NPF concentration were assayedfor trypsin biosynthesis (Borovsky et al., 1990, 1993). Fifty-fourpercent inhibition was achieved with 10⁻⁶M of NPF I indicating that NPFI affects trypsin biosynthesis in the gut by binding to a TMOF receptorand not by the release of neuroendocrine factors from the brain or thethoracic ganglia that in turn release TMOF from the ovary. Because thestructure of NPF I is different from TMOF it appears that NPF I does notbind to TMOF specific binding site on the gut receptor but to adifferent site on the same or different receptor.

EXAMPLE 2 Effect of NPF Polypeptides on Mosquito Larvae

[0091] NPF polypeptides of the subject invention have been found to behighly effective pest control agents. NPF polypeptides sharing sequencehomology with NPF I and NPF II of the Colorado potato beetle(Leptinotarsa decemlineata) (Spittaels, et al. Insect Biochem. Mol.Biol., 26(4):375-382, 1996) have been identified in the Americancockroach (Periplaneta americana) (Veenstra and Lambrou, Biochem.Biophys. Res. Commun., 213(2):519-524, 1995), mosquito (Aedes aegypti)(Stanek et al., Display Presentation, Entomological Society of AmericaAnnual Meeting, Montreal, Canada, Dec. 3-6, 2000), and fruit fly(Drosophila melanogaster), and their pesticidal activity was confirmed.Synthesized polypeptides corresponding to the native polypeptides ofColorado potato beetle (SEQ ID NO. 1), mosquito (SEQ ID NOs. 5 and 7),American cockroach (SEQ ID NOs. 9 and 11), and fruit fly (SEQ ID NOs. 13and 15) were provided to first instar Aedes aegypti larvae foringestion. Non-amidated versions of SEQ ID NOs. 5, 7, 9, 11, 13, and 15(SEQ ID NOs. 6, 8, 10, 12, 14, and 16, respectively), as well as otherfunctional equivalents (SEQ ID NOs. 17-24), were also provided foringestion.

[0092] First instar Aedes aegypti larvae were assayed in a microtiterplate in 188 μl solution containing 160 μl of water, 10 μl of 2%Brewer's yeast, and NPF polypeptides of the subject invention atconcentrations of 2 mg/ml to 0.04 mg/ml (Table 2). Mortality wasdetermined at 24 hour intervals for 3 to 7 days. Controls were run withyeast solution lacking the NPF polypeptide. TABLE 2 Effect of NPFpolypeptides on Aedes aegypti larvae LC₅₀ (mM) SEQ ID NO. NPFPolypeptide ± SEM SEQ ID NO.1Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-NH₂ 0.65 ± 0.040 (Mol.Wt.:1211.30) SEQ ID NO.5 Arg-Pro-Pro-Thr-Arg-Phe-Arg-Phe-NH₂ 0.61 ± 0.018(Mol. Wt.: 1074.23) SEQ ID NO.6 Arg-Pro-Pro-Thr-Arg-Phe-Arg-Phe-OH 0.26± 10.02 (Mol. Wt.: 1076.22) SEQ ID NO.7Ala-Pro-Gln-Leu-Arg-Leu-Arg-Phe-NH₂ 0.8 ± 0.06 (Mol. Wt.: 999.2) SEQ IDNO.8 Ala-Pro-Gln-Leu-Arg-Leu-Arg-Phe-OH 0.65 ± 0.06  (Mol Wt.: 1000.18)SEQ ID NO.9 Ala-Asn-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-NH₂ 0.135± 0.06   (Mol. Wt.: 1315.49) SEQ ID NO.10Ala-Asn-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-OH 0.68 ± 0.037 (Mol. Wt.:1316.47) SEQ ID NO.11 Ala-Asp-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-NH₂0.65 ± 0.03  (Mol. Wt.: 1315.47) SEQ ID NO.12Ala-Asp-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-OH 0.607 ± 0.066  (Mol. Wt.:1317.45) SEQ ID NO.13 Pro-Ile-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-NH₂0.589 ± 0.04   (Mol. Wt.: 1253.52) SEQ ID NO.14Pro-Ile-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-OH 0.558 ± 0.03   (Mol. Wt.:1254.5) SEQ ID NO.15 Ala-Gln-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-NH₂1.03 ± 0.1   (Mol. Wt.: 1329.52) SEQ ID NO.16Ala-Gln-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-OH 1.187 ± 0.11   (Mol. Wt.:1330.5) SEQ ID NO.17 Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-NH₂ 0.82± 0.07  (Mol. Wt.: 1130.32) SEQ ID NO.18Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-OH 0.76 ± 0.04  (Mol.Wt.: 1131.3)SEQ ID NO.19 Pro-Ser-Leu-Arg-Leu-Arg-Phe-NH₂ >2.25 (Mol. Wt.: 887.07)SEQ ID NO 20 Pro-Ser-Leu-Arg-Leu-Arg-Phe-OH 0.76 ± 0.051 (Mol. Wt.:888.05) SEQ ID NO.21 Leu-Arg-Leu-Arg-Phe-NH₂ >2.8 (Mol. Wt.: 702.89) SEQID NO.22 Leu-Arg-Leu-Arg-Phe-OH 1.589 ± 0.143 (Mol. Wt.: 703.87) SEQ IDNO.23 Arg-Pro-Pro-Thr-OH 2.08 ± 0.15 (Mol. Wt.: 469.52) SEQ ID NO.24Arg-Phe-Arg-Phe-OH 0.81 ± 0.06 (Mol. Wt.: 624.72)

EXAMPLE 3 Cytoimmunochemical Analysis

[0093] Cytoimmunochemical analysis of the mosquito gut after the bloodmeal using antiserum against NPF I revealed that exocrine cells with NPFI-like molecules are synthesized by the mosquito epithelial cells 24hours after a blood meal. In females that did not take a blood mealthese cells are not found. Thus, it is possible that NPF I is asecondary signal in a cascade of signals that starts with the release ofTMOF from the ovary, the hormone then birids to a TMOF gut receptor(Borovsky et al., 1994) that stimulates the synthesis and release of NPFI from gut specific exocrine cells. NPF I binds to a receptor NPF Ibinds to a receptor site on the gut the binding site may be adjacent toor part of the TMOF receptor and causes the cessation of trypsinbiosynthesis.

EXAMPLE 4 Bioassays for Activity Against Lepidopteron and Coleopterans

[0094] Biological activity of the pest control compounds of the subjectinvention can be confirmed using standard bioassay procedures. One suchassay is the budworm-bollworm (Heliothis virescens [Fabricius] andHelicoverpa zea [Boddie]) assay. Lepidoptera bioassays can be conductedwith either surface application to artificial insect diet or dietincorporation of samples. All Lepidopteran insects can be tested fromthe neonate stage to the second instar. All assays can be conducted witheither toasted soy flour artificial diet or black cutworm artificialdiet (BioServ, Frenchtown, N.J.).

[0095] Diet incorporation can be conducted by mixing the samplescontaining the pest-control compound with artificial diet at a rate of 6mL suspension plus 54 mL diet. After vortexing, this mixture is pouredinto plastic trays with compartmentalized 3-ml wells (Nutrend ContainerCorporation, Jacksonville, Fla.). A water blank containing no pestcontrol compound serves as the control. First instar larvae (USDA-ARS,Stoneville, Miss.) are placed onto the diet mixture. Wells are thensealed with Mylar sheeting (ClearLam Packaging, IL) using a tackingiron, and several pinholes are made in each well to provide gasexchange. Larvae can be held at 25° C. for 6 days in a 14:10(light:dark) holding room. Mortality and stunting are then recordedafter six days.

[0096] Bioassay by the top load method utilizes the same sample and dietpreparations as listed above. The samples are applied to the surface ofthe insect diet. In a specific embodiment, surface area can range from0.3 to approximately 0.8 cm² depending on the tray size; 96 well tissueculture plates can be used in addition to the format listed above.Following application, samples are allowed to air dry before insectinfestation. A water blank containing no control compound can serve asthe control. Eggs are applied to each treated well. The wells are thensealed with Mylar sheeting (ClearLam Packaging, IL) using a tackingiron, and pinholes are made in each well to provide gas exchange.Bioassays are held at 25° C. for 7 days in a 14:10 (light:dark) or 28°C. for 4 days in a 14:10 (light:dark) holding room. Mortality and insectstunting are recorded at the end of each bioassay.

[0097] Another assay useful according to the subject invention is theWestern corn rootworm assay. Samples can be bioassayed against neonatewestern corn rootworm larvae (Diabrotica virgifera virgifera) viatop-loading of the pest control sample onto an agar-based artificialdiet at a rate of 160 ml/cm². Artificial diet can be dispensed into 0.78cm² wells in 48-well tissue culture or similar plates and allowed toharden. After the diet solidifies, samples are dispensed by pipette ontothe diet surface. Excess liquid is then evaporated from the surfaceprior to transferring approximately three neonate larvae per well ontothe diet surface by camel's hair brush. To prevent insect escape whileallowing gas exchange, wells are heat-sealed with 2-mil punchedpolyester film with 27HT adhesive (Oliver Products Company, GrandRapids, Mich.). Bioassays are held in darkness at 25° C., and mortalityscored after four days.

[0098] Analogous bioassays can be performed by those skilled in the artto assess activity against other pests, such as the black cutworm(Agrotis epsilon).

EXAMPLE 5 Target Pests

[0099] Toxins of the subject invention can be used, alone or incombination with other toxins, to control one or more non-mammalianpests. These pests may be, for example, those listed in Table 3.Activity can readily be confirmed using the bioassays provided herein,adaptations of these bioassays, and/or other bioassays well known tothose skilled in the art. TABLE 3 Target pest species ORDER/Common NameLatin Name LEPIDOPTERA European Corn Borer Ostrinia nubilalis EuropeanCorn Borer resistant to Cry1A Ostrinia nubilalis Black Cutworm Agrotisipsilon Fall Armyworm Spodoptera frugiperda Southwestern Corn BorerDiatraea grandiosella Corn Earworm/Bollworm Helicoverpa zea TobaccoBudworm Heliothis virescens Tobacco Budworm Rs Heliothis virescensSunflower Head Moth Homeosoma ellectellum Banded Sunflower Moth Cochylishospes Argentine Looper Rachiplusia nu Spilosoma Spilosoma virginicaBertha Armyworm Mamestra configurata Diamondback Moth Plutellaxylostells COLEOPTERA Red Sunflower Seed Weevil Smicronyx fulvusSunflower Stem Weevil Cylindrocopturus adspersus Sunflower BeetleZygoramma exclamationis Canola Flea Beetle Phyllotreta cruciferaeWestern Corn Rootworm Diabrotica virgifera virgifera DIPTERA Hessian FlyMayetiola destructor HOMOPTERA Greenbug Schizaphis graminum HEMIPTERALygus Bug Lygus lineolaris NEMATODA Heterodera glycines

EXAMPLE 6 Insertion of Toxin Genes Into Plants

[0100] One aspect of the subject invention is the transformation ofplants with genes encoding the insecticidal toxin of the presentinvention. The transformed plants are resistant to attack by the targetpest.

[0101] Genes encoding pesticidal toxins, as disclosed herein, can beinserted into plant cells using a variety of techniques which are wellknown in the art. For example, a large number of cloning vectorscomprising a replication system in E. coli and a marker that permitsselection of the transformed cells are available for preparation for theinsertion of foreign genes into higher plants. The vectors comprise, forexample, pBR322, pUC series, M13 mp series, pACYC184, etc. Accordingly,the sequence encoding the Bacillus toxin can be inserted into the vectorat a suitable restriction site. The resulting plasmid is used totransform E. coli. The E. coli cells are cultivated in a suitablenutrient medium, then harvested and lysed. The plasmid is recovered.Sequence analysis, restriction analysis, electrophoresis, and otherbiochemical and/or molecular biological methods are generally carriedout as methods of analysis. After each manipulation, the DNA sequenceused can be cleaved and joined to the next DNA sequence. Each plasmidsequence can be cloned in the same or other plasmids. Once the insertedDNA has been integrated in the genome, it is relatively stable thereand, as a rule, does not come out again. It normally contains aselection marker that confers on the transformed plant cells resistanceto a biocide or an antibiotic, such as kanamycin, G418, bleomycin,hygromycin, or chloramphenicol, inter alia. The individually employedmarker should accordingly permit the selection of transformed cellsrather than cells that do not contain the inserted DNA.

[0102] A large number of techniques are available for inserting DNA intoa plant host cell. These techniques include transformation with T-DNA(“transferred DNA”; discussed in more detail below) using Agrobacteriumtumefaciens or Agrobacterium rhizogenes as transformation agent, fusion,injection, biolistics (microparticle bombardment), or electroporationand other methods known to those of skill in the art.

[0103] One of the most widely used approaches for the introduction ofDNA into plant cells exploits the natural DNA-transferring properties ofAgrobacterium tumefacients and Agrobacterium rhizogenes, the two specieswhich cause crown gall and hairy root. Their ability to cause diseasedepends on the presence of large plasmids, in excess of 100 kb, whichare referred to as the “Tumour-inducing” or (Ti) and “Root-inducing” (orRi) plasmids respectively.

[0104] A region referred to as the T-DNA (“Transferred DNA”) istransferred from an infecting Agrobacterium cell into the nucleus of theplant cell, where it is integrated into the plant genome. Transfer ofthe T-DNA depends on a set of genes called vir if they are on the Tiplasmid, or chv if they are on the chromosome. These genes are inducedin response to various compounds in exudates from wounded plants. TheT-DNA itself is flanked by repeated sequences of around 25 base pairs,called border repeats (or left and right borders). The T-DNA contains agroup of genes referred to as the onc genes, which are responsible forthe oncogenicity of the T-DNA.

[0105] The use of Agrobacterium in the genetic manipulation of plantsinvolves the insertion of foreign DNA into the T-DNA of a bacterial celland subsequent transfer of the DNA by the transformed bacterium into theplant. As long as the necessary proteins are provided by the bacterium,any sequences flanked by the T-DNA border repeats can be transferredinto the recipient plant cell genome. The Ti plasmids are too large tomanipulate directly, but this problem can be circumvented by usingcointegrative and binary systems.

[0106] The two main components of a cointegrative system are a Tiplasmid that has typically been modified by the replacement of materialbetween the border repeats (including the onc sequences) by pBR322; anda intermediate vector, which is a modified pBR322 containing an extramarker, such as kanamycin resistance. The gene to be introduced into thetarget plant is first cloned in to the intermediate vector, and thisconstruct is then introduced into Agrobacterium containing the Tivector. The pBR322-based plasmid cannot replicate efficiently insideAgrobacterium, so selection for kanamycin resistance identifies thoseAgrobacterium cells where the pBR322-based intermediate plasmid has beenintegrated by homologous recombination into the Ti plasmid. Because therecombination is homologous, it will take place across the pBR322sequences and therefore result in integration between the borderrepeats.

[0107] The need for cointegration of the plasmids can be circumvented byuse of a binary vector, such as pBin19, a small plasmid containing apair of left and right borders. The lacZ region, located within theborders, facilitates insertion and detection of DNA. A neomycinphosphotransferase gene, typically modified for expression in plants byaddition of nopalline synthase expression sequences, is also presentwithin the borders. Outside the left and right borders, there istypically a kanamycin resistance gene that will function in prokaryotesand a broad host-range origin derived from the plasmid pRK252. Theproteins that catalyze transfer of the T-DNA into the host plant do nothave to be cis-encoded (i.e., do not have to be encoded by the samemolecule). Therefore, if the binary vector is introduced intoAgrobacterium that already contains a resident Ti plasmid, the residentplasmid can provide all the functions needed to transfer into a plantnucleus the DNA between the borders of the binary vector.

[0108] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and the scope of the appended claims.

1. A method of controlling a pest wherein said method comprises applyingto the pest, or to a pest-inhabited locus, a pesticidally effectiveamount of an agent selected from the group consisting of: (a) NPFpolypeptides and functional equivalents thereof; (b) cells comprising apolynucleotide encoding an NPF polypeptide and/or a functionalequivalent thereof, which cells express said polynucleotide orfunctional equivalent to produce said NPF polypeptide; and (c) virusescomprising a polynucleotide encoding an NPF polypeptide, or a functionalequivalent thereof.
 2. The method, according to claim 1, wherein the NPFpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 2, and functional equivalentsthereof.
 3. The method, according to claim 1, wherein the NPFpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO. 3, SEQ ID NO. 4, and functional equivalentsthereof.
 4. The method, according to claim 1, wherein the NPFpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8,SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO.13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ IDNO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQID NO. 23, SEQ ID NO. 24, and functional equivalents thereof.
 5. Themethod, according to claim 1, wherein the NPF polypeptide is a fusionpolypeptide.
 6. The method, according to claim 1, wherein the agent is apest food cell comprising a polynucleotide encoding an NPF polypeptide.7. The method, according to claim 1, wherein the NPF polypeptide hasfrom 2 to 10 amino acid residues.
 8. The method, according to claim 1,wherein the NPF polypeptide has from 2 to 8 amino acid residues.
 9. Themethod, according to claim 1, wherein the NPF polypeptide has from 2 to5 amino acid residues.
 10. The method, according to claim 1, wherein theNPF polypeptide inhibits synthesis of insect digestive enzymes.
 11. Themethod, according to claim 1, wherein the agent comprises a dextrorotaryamino acid.
 12. The method, according to claim 1, wherein the agentcomprises a non-classical amino acid.
 13. The method, according to claim1, wherein the NPF polypeptide is N-terminal carboxylated or C-terminalamidated or both.
 14. The method, according to claim 1, wherein thepolynucleotide is optimized for expression in said cell.
 15. The method,according to claim 1, wherein the cells are mosquito larvae food cells.16. The method, according to claim 1, wherein the cell is an algae. 17.The method, according to claim 1, wherein the cell is a Clorellaspecies.
 18. The method, according to claim 1, wherein the cell is ayeast cell.
 19. The method, according to claim 1, wherein the cell isapplied in a living state.
 20. The method, according to claim 1, whereinthe cell is applied in a non-living state.
 21. The method, according toclaim 1, wherein the agent is administered as a component of apesticidal composition which also comprises a pesticidally effectivecarrier.
 22. The method, according to claim 1, wherein the pest isselected from the group consisting of coleopterans, lepidopterans, anddipterans.
 23. The method, according to claim 1, wherein the pest is ablood-sucking pest.
 24. The method, according to claim 1, wherein thepest is a pest of the suborder Nematocera.
 25. The method, according toclaim 1, wherein the pest is a pest of the family Colicidae.
 26. Themethod, according to claim 1, wherein the pest is a dipteran.
 27. Themethod, according to claim 1, wherein the pest is a pest of a genusselected from the group consisting of Heliothis, Culex, Theobaldia,Aedes, Anopheles, Forciponiyia, Culicoides and Helea.
 28. The method,according to claim 1, wherein the pest is selected from the groupconsisting of mosquitoes, flesh flies, fleas, sand flies, house flies,and dog flies.
 29. The method, according to claim 1, wherein the pest isa mosquito.
 30. The method, according to claim 1, wherein the pest is apest species selected from the group consisting of: Aedes aegypti, Culexquinquefasciatus, Anopheles albimanus, Anopheles quadrimaculatus,Lutzomyia anthrophora, Culicoides variipennis, Stomoxys calcitrans,Musca domestica, Ctenocephalides felis, and Heliothis virescens.
 31. Themethod, according to claim 1, comprising applying the agent to apest-inhabited locus.
 32. The method, according to claim 31, wherein thepest-inhabited locus is a body of water.
 33. The method, according toclaim 1, wherein the agent is administered in association with a pestfood.
 34. A method of preparing a pesticidal composition comprisingtransforming a pest food organism with a polynucleotide encoding an NPFpolypeptide and bringing said transformed pest food organism intoassociation with a pesticidally acceptable carrier.
 35. The method,according to claim 34, wherein the NPF polypeptide comprises an aminoacid sequence selected from the group consisting of SEQ ID NO. 1, SEQ IDNO. 2, and functional equivalents thereof.
 36. The method, according toclaim 34 wherein the NPF polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 4, andfunctional equivalents thereof.
 37. The method, according to claim 34wherein the NPF polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7,SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12,SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO.17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ IDNO. 22, SEQ ID NO. 23, SEQ ID NO. 24, and functional equivalentsthereof.
 38. The method, according to claim 34, wherein the NPFpolypeptide is produced in the pest food organism as a fusionpolypeptide.
 39. The method, according to claim 34, wherein the NPFpolypeptide inhibits synthesis of insect digestive enzymes.
 40. Themethod, according to claim 34, wherein the polynucleotide is optimizedfor expression in said pest food organism.
 41. The method, according toclaim 34, wherein the NPF polypeptide is N-terminal carboxylated orC-terminal amidated or both.
 42. The method, according to claim 34,wherein the NPF polypeptide comprises a dextrorotary amino acid.
 43. Themethod, according to claim 34, wherein the NPF polypeptide comprises anon-classical amino acid.
 44. An expression vector comprising a promoterand a polynucleotide encoding an NPF polypeptide wherein the promoterhas the capacity to control expression of the NPF polypeptide in a foodorganism of a pest.
 45. The expression vector, according to claim 44,wherein the NPF polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, and functionalequivalents thereof.
 46. The expression vector, according to claim 44,wherein the NPF polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO. 3, SEQ ID NO. 4, and functionalequivalents thereof.
 47. The expression vector, according to claim 44,wherein the NPF polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7,SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12,SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO.17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ IDNO. 22, SEQ ID NO. 23, SEQ ID NO. 24, and functional equivalentsthereof.
 48. The expression vector, according to claim 44, wherein theNPF polypeptide is a fusion polypeptide.
 49. The expression vector,according to claim 44, wherein the polynucleotide is optimized forexpression in said organism.
 50. A transformed cell comprising apolynucleotide encoding an NPF polypeptide, which cell expresses saidpolynucleotide to produce said NPF polypeptide.
 51. The transformedcell, according to claim 50, wherein the NPF polypeptide comprises anamino acid sequence selected from the group consisting of SEQ ID NO. 1,SEQ ID NO. 2, and functional equivalents thereof.
 52. The transformedcell, according to claim 50, wherein the NPF polypeptide comprises anamino acid sequence selected from the group consisting of SEQ ID NO. 3,SEQ ID NO. 4, and functional equivalents thereof.
 53. The transformedcell, according to claim 50, wherein the NPF polypeptide comprises anamino acid sequence selected from the group consisting of SEQ ID NO. 5,SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10,SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO.15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ IDNO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, andfunctional equivalents thereof.
 54. The transformed cell, according toclaim 50, wherein the NPF polypeptide inhibits synthesis of an insectdigestive enzyme.
 55. The transformed cell, according to claim 50,wherein the digestive enzyme is selected from the group consisting oftrypsin and trypsin-like enzyme.
 56. The transformed cell, according toclaim 50, wherein the transformed cell is a pest food.
 57. Thetransformed cell, according to claim 50, wherein the transformed cell ismosquito larvae food.
 58. The transformed cell, according to claim 50,wherein the transformed cell is a green algae.
 59. The transformed cell,according to claim 50, wherein the transformed cell is a Clorellaspecies.
 60. The transformed cell, according to claim 50, wherein thetransformed cell is a yeast cell.
 61. A pesticidal compositioncomprising: (a) an agent selected from the group consisting of: NPFpolypeptides and functional equivalents thereof, pest food cellscomprising a polynucleotide encoding an NPF polypeptide and/or encodingfunctional equivalents of an NPF polypeptide; and viruses comprising apolynucleotide encoding an NPF polypeptide, or encoding functionalequivalents of an NPF polypeptide; together with (b) a pesticidallyacceptable carrier.
 62. The pesticidal composition, according to claim61, wherein the NPF polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, andfunctional equivalents thereof.
 63. The pesticidal composition,according to claim 61, wherein the NPF polypeptide comprises an aminoacid sequence selected from the group consisting of SEQ ID NO. 3, SEQ IDNO. 4, and functional equivalents thereof.
 64. The pesticidalcomposition, according to claim 61, wherein the NPF polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9,SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO.14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ IDNO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQID NO. 24, and functional equivalents thereof.
 65. The pesticidalcomposition, according to claim 61, wherein said polypeptide is a fusionpolypeptide.
 66. The pesticidal composition, according to claim 61,formulated as a slow-release formula.
 67. The pesticidal composition,according to claim 61, in a form selected from the group consisting ofpellets, briquettes, bricks, powders, granules, sprays, solutions andcapsules.
 68. The pesticidal composition, according to claim 61,formulated to float on an aqueous medium.
 69. The pesticidalcomposition, according to claim 61, formulated to maintain a depth of 0to 2 feet below the surface of an aqueous medium.
 70. The pesticidalcomposition, according to claim 61, formulated to sink in an aqueousmedium.